Mapping the genomic organization of modified histones and defining locations of DNA binding proteins is critical for deep understanding of the regulatory mechanisms governing cellular states. However, the primary methodology used for mapping DNA associated proteins ? chromatin immunoprecipitation followed by sequencing (ChIP-seq) ? has several major limitations: (i) standard ChIP-seq is unable to profile more than one epitope at a time in one sample; (ii) the immunoprecipitation step is inefficient, resulting in low signal to noise ratios; and (iii) ChIP-seq signal is effectively an average readout from large populations of cells due to input material requirements. Thus, our challenge is to simultaneously study the organization of multiple DNA binding proteins from a single sample. Our proposed project will develop a novel methodology termed `Whip- seq' that is designed to overcome these limitations, in part by avoiding immunoprecipitation altogether through the use of long specialized oligonucleotides and DNA barcoding. The Whip-seq method aspires to provide a simple, rapid, low-cost means to address many to date unanswerable questions in chromatin biology. In the experiments outlined in this proposal, we will: Develop the Whip-seq method in a stepwise manner in vitro (Aim1); implement a computational framework for data analysis (Aim 2); and establish the performance of the new assay using in vivo mouse ES cells (Aim 3). Whip-seq has the potential to transform the mapping of chromatin into a highly sensitive, cheap and robust process that can be carried out in any molecular biology laboratory. Whip-seq will provide a means to obtain an unprecedented view of the different combinatorial signatures of chromatin states and how these signatures may be dysregulated in disease, e.g., cancer. The high-resolution mapping, and the ease of implementation, may enable Whip-seq to serve as a diagnostic tool.